Plasma membrane protein (sometimes referred to as membrane protein) is present as a protein constituting a biological membrane. A plasma membrane protein has a function as a receptor of an enzyme, peptide hormone, growth factor, autacoid, etc.; transporter of sugar, etc.; ion channel, or plasma membrane antigen, and is associated with dynamic functions of a cell such as penetration/transportation of a substance, by receiving a signal from the surface of a cell. A plasma membrane protein is a protein or a glycoprotein incorporated in a plasma membrane lipid bilayer, which is present in various forms, for example, penetrating all layers of a plasma membrane (transmembrane protein), located on the surface layer (cell surface protein), or undercoating a plasma membrane. In either case, it is a high molecular protein.
As one of plasma membrane protein, ABCG2 (ATP binding cassette transporter G2) is known. ABCG2 is known as a member of human ATP binding cassette (ABC) transporter, and it is reported that ABC transporter is associated with causes of diseases or drug transport. ABC transporter is one of the biggest superfamily of protein which is found in any cell of from bacterium to higher animal such as human, and about 250 members (about 50 in human) are known. Almost all ABC proteins function as a transporter, while some function as an ion channel. ABCG2 gene has been first isolated by Allikmets et al (Humann Mol. Genet. 5(10), 1649-1655, 1996). This protein is a transporter being the cause of drug resistance, and has been revealed to have a function to discharge anticancer agents to the outside of the cell (Proc. Natl. Acad. Sci., 95, 15665-15670, 1998; Clin. Cancer. Res. 7, 935-941, 2001).
Further, also as one of plasma membrane proteins, P-glycoprotein (PGP; also known as multidrug transporter MDR1) is known (Medicine, Stanford University School of Medicine, Stanford, Calif. 94306, USA; Nature 323(6090), 728-731, 1986; Biochem. Biophys. Res. Commun. 162(1), 224-231, 1989). P-glycoprotein is a member of the ABC transporter superfamily, and is expressed in human intestines, liver or other tissues. The enzyme is incorporated in the plasma membrane and acts, as an efflux pump transporting small molecules. It has been known these last several years that expression of a high-level P-glycoprotein is a mechanism of tumor resistance against chemotherapy of cancer. Further, expression of P-glycoprotein in intestines, affects oral bioavailability of an agent molecule which is a substrate of this transporter, enables effective discharge of drug in intestine lumen, thus reducing the drug amount to circulate.
Further, ABCC4 is known as one of plasma membrane proteins (Genetics, 123(1), 45-54, 1989). ABCC4 is known as a member of ATP binding domain (ABC) transporter. ABC transporter superfamily is one of the biggest gene families, which encodes membrane protein groups having various functions related to energy-dependent transportation of a wide variety of substances passing through the membrane (Curr. Opin. Genet. Dev., 5, 779-85, 1995). ABC proteins are associated with extracellular and intracellular membrane transport of various substances, including ion, amino acid, peptide, sugar, vitamin, and steroid hormone. Human ABCC subfamily has at present 10 identified members (ABCC 1-10), among which 7 are derived from multidrug resistance-like (MRP) subgroup, 2 are derived from sulfonyaurea receptor (SUR) subgroup, and 1 is CFTR gene. MRP-like protein is an organic anion transporter. ABCC4 and ABCC5 proteins are known to impart resistance against nucleotide analog including PMEA and purine base analog.
As for biogenic proteins such as the above-mentioned plasma membrane protein, it is necessary to detect proteins contained in the cells or the like, and measure them quantitatively in order to elucidate the function of the protein, or when testing or screening for the protein, or to use the protein. Particularly, when developing a novel drug, it is significant to quantify absolutely proteins such as membrane proteins to test a novel drug candidate, with a simple and accurate method.
For example, more than 90% of substances: as novel drug candidates, which pharmaceuticals companies have developed by spending much time and cost, are dropped out (halt development). Most of the reasons are drug kinetics (absorption, distribution, metabolism, excretion) such as low-absorption to digestive tracts, or drug transfer into inappropriate organs, and side effects. As drug kinetics are considered by using animals conventionally, it cannot be considered at an early phase of the development as high throughput screening. Further, as there are species differences of drug kinetics, some data obtained from animal experiments are not reproduced in human. In other words, the problem of drug kinetics is often revealed in clinical experiments at a late stage of the development, which cause significant losses for pharmaceutical companies. Due to such problems, it is awaited that human drug kinetics can be estimated at an early phase of development by using a system that enables high throughput screening in vitro.
Performance of the above system is actualized for metabolisms of drug kinetics. In other words, the absolute amount of each metabolic enzyme in human liver is revealed. Further, the metabolic velocity per molecule by each metabolic enzyme of the subject drug in vitro is calculated by using a recombinant protein. From the information from each metabolic enzyme functioning mainly in liver, the metabolic velocity of the subject drug can be estimated by calculation. In other words, a means for obtaining the absolute amount of enzyme protein in organs and in vitro is indispensable.
On the other hand, a membrane protein associated with drug kinetics (drug transporter, receptor), which transports drugs is playing a crucial role for absorption, distribution and excretion among the drug kinetics. Therefore, if the absolute amount of the membrane protein associated with drug kinetics in organs, and the transportation ability or the binding ability of the drug per molecule can be measured, it would be possible to estimate the metabolic velocity from the obtained information by calculation, similarly with metabolism. Presently, there is a system to estimate in vitro the transportation or binding by using a membrane protein forcibly expressing cell or an expressed membrane vesicle, while it is very difficult to quantify the absolute amount of membrane protein. Therefore, if a more general method for quantifying the absolute amount of membrane protein with a high sensitivity is established, it becomes possible to estimate drug kinetics from an experiment system in vitro. Thus, it would be possible to estimate drug kinetics in human at an early stage of drug development, and reduce significantly time and cost necessary for drug development. The development of a method for absolutely quantifying membrane protein is awaited also even in the field of drug development.
Conventionally, a method using electrophoresis such as two-dimensional electrophoresis was conducted for quantifying biogenic proteins. With this method, detection and quantification were performed by staining the protein to be quantified, or by autoradiography, or by using an antibody specific to a particular protein (western blot). Particularly, a method using an antibody was conducted for quantifying a plasma membrane protein. For a method for quantifying a plasma membrane protein using an antibody, it is necessary to prepare an antibody against the protein, and it cannot be applied to a membrane protein against which an antibody cannot be prepared. Further, the method for quantifying comprises solubilizing the membrane protein when preparing a sample, and then electrophoresing to stain the antibody. However, as solubilizing conditions vary upon membrane proteins, conditions must be considered individually, which takes time. Further, as it is not possible to electrophorese insoluble proteins or high-molecular proteins, it was difficult to apply these methods for quantifying a plasma membrane protein.
On the other hand, mass spectrometry is making a significant progress recently, and the method is considered and used for detecting or measuring various biological materials. Mass spectrometers having various function have been developed including: a mass spectrometer having an electrospray ionization (ESI) source, a mass spectrometer having a liquid chromatography spectrometry (LC-MS), a MS/MS spectrum or tandem mass spectrum (tandemu MS) mass spectrometer wherein two mass spectrometers are bound. They are used for detecting or measuring biological materials (Japanese Laid-Open Patent Application No. 2004-28993; Japanese Laid-Open Patent Application No. 2004-77276; Published Japanese translation of PCT international publication No. 2004-533610).
Recently, a mass spectrometry using a stable-isotope label has been developed, and is used for detecting or measuring biological materials. This method quantifies proteins or peptides in a sample by mass spectrometry, by using proteins or peptides labeled with a stable-isotope. Examples of using the method for quantifying C-reactive protein (CRP) which is a diagnostic marker in the serum of a patient with rheumatism, and -amyloid in a sample from mammalian tissues or in body fluid have been reported. However, no example of using such mass spectrometry for quantifying a plasma membrane protein which is insoluble and high-molecular is known.
Conventionally, LS/MS/MS is used for detecting or measuring biological materials by using mass spectrometry. Quantification by using LS/MS/MS is performed by using channel in which MS spectrum of the intended compound, and a particular MSMS spectrum peak are combined. For a low-molecular weight compound, usually the method is performed by using a single channel. However, in case of a peptide, which is high molecular, it was estimated that quantification using a single channel would cause some problems, and quantification using a single channel in fact caused some problems.
In other words, when there is a peptide with a different sequence from that of the selected peptide detected in the same channel, plural peaks are detected in the selected channel. It is possible to synthesize an internal standard peptide, and to identify the peak of the elution time identical with the internal standard peptide, as a peak of the intended peptide. However, when plural channels are prepared for various membrane proteins, it is very difficult to synthesize internal standard peptides for all of them, from the view points of time and cost. Therefore, a means for identifying the peak of the intended peptide without an internal standard peptide is necessary. Further, it is possible that a peptide with a different sequence detected in the same channel is detected within the same elution time as the intended peptide, which lowers quantification accuracy.
Patent reference 1: Japanese Laid-Open Patent Application No. 2004-28993
Patent reference 2: Japanese Laid-Open Patent Application. No. 2004-77276
Patent reference 3: Japanese; Laid-Open Patent Application No. 2004-157124
Patent reference 4: Published Japanese translation of PCT International Publication NO. 2004-533610
Non-patent reference 1: Humann Mol. Genet. 5(10), 1649-1655, 1996
Non-patent reference 2: Proc. Natl. Acad. Sci., 95, 15665-15670, 1998
Non-patent reference 3: Clin. Cancer. Res. 7, 935-941, 2001
Non-patent reference 4: Medicine, Stanford University School of Medicine, Stanford, Calif. 94306, USA
Non-patent reference 5: Nature 323(6090), 728-731, 1986
Non-patent reference 6: Biochem. Biophys. Res. Commun. 162(1), 224-231,
Non-patent reference 7: Genetics, 123(1), 45-54, 1989
Non-patent reference 8: Curr. Opin. Genet. Dev., 5, 779-85, 1995
Non-patent reference 9: Proteomics 4, 1175-1186, 2004